CN108359680A - Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL146 genes and its application - Google Patents
Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL146 genes and its application Download PDFInfo
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- CN108359680A CN108359680A CN201810108127.3A CN201810108127A CN108359680A CN 108359680 A CN108359680 A CN 108359680A CN 201810108127 A CN201810108127 A CN 201810108127A CN 108359680 A CN108359680 A CN 108359680A
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- human cytomegalovirus
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- AFWXOGHZEKARFH-ACRUOGEOSA-N Tyr-Tyr-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=C(O)C=C1 AFWXOGHZEKARFH-ACRUOGEOSA-N 0.000 description 1
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Abstract
The invention discloses a kind of recombinant plasmid containing human cytomegalovirus UL146 genes, genetic engineering bacterium and its applications.The recombinant plasmid is by the way that the UL146 genes of human cytomegalovirus to be inserted into escherichia coli cloning expression vector pET32a (+), obtain recombinant vector pET32a (+) UL146, then it is transformed into prokaryotic expression engineering bacteria BL21 (DE3), obtains the genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids.Corresponding UL146 recombinant proteins are obtained using the engineering bacterium expression, the fusion protein can be used for preparing the antibody for HCMV UL146 genes, and can be used for studying the function of UL146 genes, the function and its molecular mechanism for further research UL146 provide the foundation.
Description
Technical field
The present invention relates to field of biology, more particularly to a kind of recombination matter containing human cytomegalovirus UL146 genes
Grain, genetic engineering bacterium and its application.
Background technology
UL146 genes are an open reading frame (ORF) in human cytomegalovirus (HCMV), human cytomegalovirus
Genome is maximum genome in herpesviral, contains more than 200 a reading frames, and the most gene in genome is all high
What degree was guarded, but there is also the genes of high polymorphism, i.e. its gene and nucleic acid sequence is variant between different virus strain
Property, they encode some embrane-associated proteins and secretory protein such as gB, UL144, UL146 and UL147 gene.
HCMV UL146 genes are about 345~360bp, and UL146 genes are present in a height in HCMV genomes and become
The initiation site in different region, region UL146 translations starts and before through the translation initiation position of the UL147 adjoined therewith
Point, since the variation of in most cases nucleotide is non-synonymous, the variability of this gene order determines its ammonia
The polymorphism of base acid sequence, UL146 amino acid sequence height in each clinical strain of HCMV makes a variation, but there is also a small number of height
Conservative amino acid.Arav-Boger et al. has found that variations of the UL146 in different HCMV Strain is by Phylogenetic Analysis
Occurring at random, it is happened in UL146 whole genes, although the variation of ULL146 occurs at random in whole gene,
It is to occur that the amino acid sequence (ELR) of chemotactic factor (CF) functional areas is really highly conserved, this illustrates the gene for HCMV at it
It is highly important.
About 115~120 amino acid of the ORF of HCMV UL146, the small molecular protein of coding size about 13KD, Penfold
Et al. by sequence alignment analysis and chemotaxis experiment find from Toledo chemotactic factor (CF) vCXCL-1 and host cell
Factor interleukin 8 (IL-8) sequence is similar, and with chemotactic factor (CF) characteristic structural such as signal peptide, Cys residues and
Chemotactic factor (CF) combined with chemokine receptors and neutrophil activation necessary to ELR motifs, while also have chemotactic because
Certain functions of son such as induce calcium current lead to, the threshing function of the chemotaxis of inducing neutrophil and neutrophil cell,
Therefore, it is considered that UL146 codings be a class IL-8 viral chemokine, it be first viral α chemotactic being found because
Sub- vCXCL-1.Chemotactic factor (CF) is the small molecular protein that a group participates in immunoregulation, and leucocyte can be attracted to reach infection site.
According to the difference of its structure, it is classified as four classes：C, CC, CXC and CX3C.HCMV can be by being combined with chemotactic factor (CF) or being expressed
Chemotactic factor (CF) class analog albumen inhibits its function.Such as：US28 inhibits the immune of host to answer by being combined with CC chemotactic factor (CF)s
It answers；UL128 is that (a member in (gH/gL/UL128-UL130-UL131A) encodes a kind of CC chemotactic factor (CF)s to pentamer compound
And influence tissue tropism.VCXCL-1 is by the HCMV UL146 gene codes with high polymorphism, and this variation is mostly
Missense mutation, therefore the vCXCL-1 of difference HCMV Strain has otherness.It is different that Heo et al. carries out research discovery
The migration of vCXCL-1 its function as caused neutrophil cell, the affinity combined with neutrophil surface receptor and
VCXCL-1 illustrates that the polymorphism of vCXCL-1 affects vCXCL-1 and combined with receptor to the difference such as response of signal path
Affinity and neutrophil cell activation.CXCR1 can be expressed with CXCR2 receptors in neutrophil surface, be normal
The chemokine receptors seen, the two amino acid sequence have 77% homogeneity, Lu ¨ ttichau researchs to find that vCXCL-1 not only may be used
To be combined the migration that can be also combined and be caused neutrophil cell with CXCR1 with CXCR2, but combined with CXCR1 affine
Power is relatively low.Yamin et al. has found that vCXCL-1 can not only be combined with CXCR1 and CXCR2 by research, moreover it is possible to natural kill
(NK) CX3CR1 of cell surface expression is combined, and can by is combined preferential attraction neutrophil cell with CXCR2 rather than
The NK cells to play an important role in immunologic process.
It is particularly significant that the above research all shows that HCMV UL146 genes have the diffusion in vivo and propagation of HCMV
Effect, so further research UL146 effect molecular mechanism be very necessary.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of containing human cytomegalovirus disease
The recombinant plasmid of malicious UL146 genes.
Another object of the present invention is to provide the genes containing above-mentioned human cytomegalovirus UL146 gene recombination plasmids
Engineering bacteria.
Another object of the present invention is to provide the above-mentioned gene work containing cytomegalovirus UL146 gene recombination plasmids
The application of journey bacterium.
The purpose of the invention is achieved by the following technical solution：A kind of recombination containing human cytomegalovirus UL146 genes
Plasmid contains UL146 genetic fragments, wherein the nucleotide sequence of UL146 genetic fragments such as SEQ ID No：Shown in 1.
The construction method of the recombinant plasmid containing human cytomegalovirus UL146 genes by conventional method will
UL146 genetic fragments are inserted into pET32a (+) carrier and obtain.
A kind of genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids, conversion have above-mentioned huge containing someone
The recombinant plasmid of cell virus UL146 genes.
The genetic engineering bacterium is preferably Escherichia coli；More preferably bacillus coli DH 5 alpha or BL21 (DE3).
The construction method of the genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids, will be above-mentioned
Recombinant plasmid transformed containing human cytomegalovirus UL146 genes enters in prokaryotic expression engineering bacteria, obtains containing human cytomegalovirus disease
The genetic engineering bacterium of malicious UL146 gene recombination plasmids；Its construction method preferably comprises following steps：
(1) it is directed to the primer sequence of UL146 genes using the libraries BAC Towne HCMV as stencil design, introduces SalI
With two restriction enzyme sites of BamHI；
(2) using the libraries BAC Towne HCMV as template PCR amplifications UL146 genes；
(3) electrophoresis is carried out to the UL146 genes that amplification obtains in step (2) and recycles UL146DNA；
(4) UL146DNA and vector plasmid pET32a (+) that are recycled in step (3) are subjected to the bis- enzymes of SalI and BamHI
It cuts, and is purified；
(5) product in step (4) after purification is attached, makes UL146 target gene and vector plasmid pET32a
(+) is connected；
(6) it by the product conversion competent escherichia coli cell after connection in (5), obtains containing human cytomegalovirus
The genetic engineering bacterium of UL146 gene recombination plasmids.
Electrophoresis described in step (3) is agarose gel electrophoresis.
The construction method of the genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids, in step
(5) further include the steps that obtained recombinant plasmid carries out double digestion identification positive colony and is sequenced after.
The genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids is in UL146 recombinant protein systems
Application in standby.
A kind of preparation method of UL146 recombinant proteins contains human cytomegalovirus UL146 gene recombination plasmids by above-mentioned
Genetic engineering bacterium pass through activation culture and fermented and cultured, add derivant IPTG and carry out induced expression, obtain UL146 weights
Histone；Preferably：Above-mentioned engineering bacteria is seeded in the LB liquid medium containing ampicillin (Amp) resistance and is carried out
Then activation culture is transferred to containing fermented and cultured is carried out in amicillin resistance LB liquid medium, adds derivant
IPTG carries out induced expression, obtains UL146 recombinant proteins.
The condition of the induced expression is preferably：10h is induced at 20 DEG C.
The working concentration of the IPTG is 0.5mM.
The preparation method of the UL146 recombinant proteins further includes purifying the UL146 recombinant proteins of acquisition
Step.
The purifying is to be purified using affinity chromatography, and histidine tag is purification tag.
The preparation method of the UL146 recombinant proteins, further include the UL146 recombinant proteins of acquisition are carried out it is quantitative
Step：Protein quantification is carried out using BCA methods.
The genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids is preparing human cytomegalovirus disease
Application in malicious UL146 protein polyclone antibodies；The engineering bacteria can give expression to the fusion protein with histidine tag, can
Polyclonal antibody is prepared using affinitive layer purification.
Recombinant protein histidine tag containing 6 × His.
The present invention has the following advantages and effects with respect to the prior art：
1, it the purpose of the present invention is building a kind of recombinant plasmid engineering bacteria containing human cytomegalovirus UL146 genes, uses
In the molecular mechanism that research diffusions at HCMV and is propagated, basis is provided for HCMV therapy targets.
2, the present invention provides a kind of engineering bacteria including human cytomegalovirus UL146 gene recombination plasmids, by by people
The UL146 protein gene of cytomegalovirus is inserted into escherichia coli cloning expression vector pET32a (+), and recombinant vector is obtained
Then pET32a (+)-UL146 is transformed into prokaryotic expression engineering bacteria BL21 (DE3), expression of recombinant plasmid bacterium is obtained
BL21 (DE3)-pET32a (+)-UL146, and expression of the recombinant protein in BL21 (DE3) is further studied, and
Condition optimizing has been carried out to expression quantity of the recombinant protein in protokaryon；Recycle the weight of Western blot methods identification induction
Histone and the method for using affinity chromatography isolate and purify the recombinant protein with His labels；Finally BCA is used to send out
The recombinant protein of purifying is quantified.The antibody for UL146 genes can be prepared on the basis of the present invention or is used for
The function of in vitro study UL146.
Description of the drawings
Fig. 1 is pET32a (+)-UL146 construction of recombinant plasmid schematic diagrames.
Fig. 2 is UL146 gene magnification result figures；Wherein, swimming lane M is DNA marker, and swimming lane 1 is UL146 PCR amplifications
Product.
Fig. 3 is the double digestion result figure of pET32a (+)-UL146 recombinant expression carriers；Wherein, swimming lane M is DNA
marker；Swimming lane 1 is pET32a (+)-UL146 recombinant expression carriers through restriction enzyme SalI and BamHI double digestion；Swimming
Road 2 is pET32a (+)-UL146 recombinant expression carriers without digestion.
Fig. 4 is the temperature optimization result figure of SDS-PAGE analysis recombinant protein protokaryon induced expressions；Wherein, swimming lane M is egg
White marker；Swimming lane 1,3,5 does not add the expression of each albumen in BL21 (DE3) before derivant when being respectively 20 DEG C, 30 DEG C, 37 DEG C
Situation；Swimming lane 2,4,6 adds the expression of BL21 (DE3) each albumen after derivant when being respectively 20 DEG C, 30 DEG C, 37 DEG C.
Fig. 5 is the IPTG concentration optimization result figures of SDS-PAGE analysis recombinant protein protokaryon induced expressions；Wherein, swimming lane M
For albumen marker；Swimming lane 1,2,3 and 4 is respectively that addition IPTG concentration is respectively 0.2Mm, 0.5mM, 1.0Mm, 2.0mM inductions
Afterwards in BL21 (DE3) bacterial strain albumen expression；5,6,7,8 swimming lane of swimming lane is respectively that BL21 (DE3) bacterium before derivant is added
The expression of albumen in strain.
Fig. 6 is the time-optimized result figure of SDS-PAGE analysis recombinant protein protokaryon induced expressions；Wherein swimming lane M is
Albumen marker；Swimming lane 1~9 be respectively induce 0h, 2h, 4h, 5h, 6h, 8h, 10h, 12h, for 24 hours when BL21 (DE3) bacterial strain in
The expression of albumen.
Fig. 7 is the result figure in the expression place of SDS-PAGE analysis recombinant proteins；Wherein swimming lane M is albumen marker；Swimming
Road 1 and 2 is culture supernatant；Swimming lane 3 and 4 is cell pyrolysis liquid；Swimming lane 5 and 6 is soluble protein, and swimming lane 7 and 8 is to refer to forgive
Body；Swimming lane 1,3,5,7 is the expression of albumen in BL21 (DE3) bacterial strain induced without IPTG；Swimming lane 2,4,6,8 lures for IPTG
The expression of albumen in BL21 (DE3) bacterial strain led.
Fig. 8 is the result figure in the expression place of Western blot identification recombinant proteins.
Fig. 9 is the purification result figure of the recombinant protein of SDS-PAGE analyses after purification；Wherein, swimming lane M is albumen
Marker, swimming lane 1 are to forgive liquid solution；Swimming lane 2 and 3 is to be pierced by albumen；Swimming lane 4 is albumen after purification.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
One, construction of recombinant plasmid
(1) UL146 gene primers design
According to the gene order (GenBank for the HCMV BAC towne being published on genebank:
FJ616285.1), PCR primer is designed through Primer Primier 5.0.The primer length of amplification is 357bp (nucleotide sequences
Such as SEQ ID No：Shown in 1, protein translation product such as SEQ ID No：Shown in 2).
Designed primer such as following table：
1 UL146 design of primers tables of table
Note：F (sense primer)；R (downstream primer)；It is corresponding restriction enzyme site at underscore.
(2) UL146 gene PCRs
With the libraries HCMV BAC Towne (bibliography：Human embryonic lung fibroblasts
treated with artesunate exhibit reduced rates of proliferation and human
Cytomegalovirus infection in vitro) be PCR template, PCR is carried out in following reaction system：
2 PCR reaction system tables of table
| Component | Volume (μ L) |
| 5×Primer STAR Buffer | 10 |
| dNTP Mixture | 4 |
| Sense primer | 2 |
| Downstream primer | 2 |
| Template (Template) | 1 |
| Primer STAR HS DNA polymerase | 0.5 |
| ddH2O | 30.5 |
| Total volume | 50 |
Table is arranged in 3 PCR reaction conditions of table
(3) PCR product is through 2% agarose gel analysis, and passes through plastic recovery kit Gel Extraction kit
(OMEGA Bio-Tek, the U.S.) recycles PCR product.
PCR product recycling step is as follows：
1. PCR product after electrophoresis, cuts the gel containing purpose band under the conditions of ultraviolet and is placed in 1.5 clean mL's
In EP pipes；
2. being added and the isometric XP2 solution of gel in EP pipes；
3. the EP pipes of the 1.5mL for the gel piece for being mixed with XP2 solution are placed in water-bath, 57 DEG C of heating, every 2~
3min overturns mixing, until gel piece melts completely；
4. its mediation mixing and of short duration centrifugation, can finally be added into advance by the gel cooling after complete melt
Assembled silicagel column, room temperature, 10000 × g centrifuge 1min, topple over the waste liquid in collecting pipe；
5. it adds in 300 μ L XP2 solution to silicagel column, it, will be in collecting pipe after room temperature 10000 × g centrifugations 1min
Liquid is outwelled；
6. washing solution with 700 μ L SPW washs silicagel column, 10000 × g of room temperature centrifuges 1min, by the liquid in collecting pipe
Body is outwelled；
7. it is primary to repeat 6 steps；
8. sky gets rid of silicagel column, 12000 × g of room temperature centrifuges 2min, removes extra SPW washing solution；
9. taking out silicagel column and putting it into the 1.5mL EP pipes of sterilizing, after being placed at room temperature for 2min, into silicagel column
30 μ L are added and have been heated to 65 DEG C of sterile ddH20 in advance, after being stored at room temperature 1min, 12000 × g centrifuges 1min, and taking-up has been discarded
Silicagel column, the liquid collected in EP pipes is target DNA solution；
10. detecting the target DNA concentration and purity of recycling, sample is put in -20 DEG C of preservations.
Experimental result：As shown in Fig. 2, consistent with expected size result, target gene fragment size is pcr amplification product
357bp。
(4) plasmid extraction
1. taking out the plasmid evening before yesterday, from -20 DEG C of refrigerators taking-ups, containing pET32a (+) plasmid, (plasmid is purchased from the excellent precious limited public affairs of biology
Department) DH5 α strains, draw 40 μ L, be inoculated in the 4mL LB containing ampicillin (ampicillin concentration be 100 μ g/mL)
In fluid nutrient medium, 250rpm/min cultivates 12~16h in 37 DEG C of incubators；
2. second day, taking out the bacterium solution being incubated overnight, and thalline is added in 1.5mL EP pipes, 12000 × g room temperatures
1min is centrifuged, supernatant is abandoned, step is repeated, 5mL bacterium solutions is all collected into 1.5mL EP pipes；
3. after the extra supernatant that exhausts, adding in 250 μ L solution I to EP pipes, it is heavy that thalline is gently blown and beaten with pipettor
It forms sediment, after thalline resuspension, is put on the rotary vibrator of whirlpool, makes its complete mixing；
4. adding 250 μ L solution II, for several times, the reverse time will be got hold of the mixing that lightly turns upside down, control
System is between 2min~5min, and after having overturned, liquid generally can transparent clarification；
5. be eventually adding 350 μ L solution III, the mixing that lightly turns upside down for several times, until there is white precipitate shape
At overturning mixing number cannot be excessive, is and then put in compact centrifuge, room temperature, and 13000 × g centrifuges 10min；
6. after centrifugation, liquid-transfering gun is transferred to supernatant in marked good silicagel column, silicagel column puts 2mL into
Centrifuge tube in, not by white depositions suck silicagel column in, be put in compact centrifuge, 10000 × g of room temperature, centrifuge
1min；
7. outwell the waste liquid in collecting pipe, draw 500 μ L HB solution, be added in silicagel column, 10000 × g of room temperature from
Heart 1min；
8. outwelling the waste liquid in collecting pipe, adds in 700 μ L DNA Wash Buffer to silicagel column, (please first check DNA
Whether Wash Buffer have been added absolute ethyl alcohol！) room temperature 10000 × g centrifugations 1min；
9. it is primary to repeat 8 steps；
10. sky gets rid of silicagel column, 13000 × g of room temperature centrifuges 2min, removes extra DNA Wash Buffer；
It takes out silicagel column to put it into the 1.5mL EP pipes of sterilizing, the 30 μ L things of μ l~50 is added into silicagel column
65 DEG C of sterile ddH20 first are heated to, after being stored at room temperature 1~2min, 12000 × g centrifuges 1min, takes out obsolete silica gel
Column, liquid is target DNA solution in EP pipes；
The detection of 1 μ L samples is taken to extract the concentration and purity of plasmid, remaining sample is for subsequent experimental or is put in -20
DEG C preserve.
(5) double digestion
The plasmid extracted and the PCR product of recycling are subjected to double digestion, double digestion body in 37 DEG C of water bath with thermostatic control 40min
System such as following table：
4 PCR product double digestion system table of table
Completing plasmid and PCR product after endonuclease reaction need to be purified, with Product Purification Kit recycling double digestion production
Object, purification step are as follows：
1. 4~5 times of combination liquid CP is added according to double digestion system, mix well；
2. previous step acquired solution is added in adsorption column EC, it is placed at room temperature for 1min, 10000 × g, 1 min of centrifugation is outwelled
Waste liquid in collecting pipe；
3. 700 μ L rinsing liquid Wash Buffer (first check whether and absolute ethyl alcohol has been added), 10000 × g are added, centrifuge
1min discards waste liquid；
4. it is primary to repeat 3 steps；
5. adsorption column EC is put back in collecting pipe, 10000 × g, 1min is centrifuged, removes rinsing liquid as far as possible, in order to avoid residual
Ethyl alcohol inhibits downstream reaction；
6. taking out adsorption column EC, it is put into a clean centrifuge tube, adds 30~50 μ L to wash at the intermediate position of adsorbed film
De- buffer solution EB (heating effect is more preferable in 65~70 DEG C of water-baths in advance for elution buffer), is placed at room temperature for 2min, 13000 × g
Centrifuge 1min.If necessary to more amount DNA, obtained solution can be rejoined in adsorption column, centrifuge 1min.Taking-up has been given up
The silicagel column abandoned, the liquid write down notes in EP pipes is target DNA solution；
7. the target DNA concentration and purity of recycling are detected, to be preferably used for subsequent experimental.Sample is put in -20 DEG C of guarantors
It deposits.
(6) enzyme connects
Plasmid and target DNA after double digestion is recycled connect 16h or more at 16 DEG C, and enzyme coupled reaction system is as follows：
5 coupled reaction system table of table
(7) preparation of bacillus coli DH 5 alpha competent cell
1. DH5 α competence previous evening is done, from -20 DEG C of taking-up zero load DH5 α strains, by volume 1：100 are inoculated with,
37 DEG C of constant-temperature table 250rpm/min activate 12~16h；
2. second day, again by volume 1 by activated DH5 α empty bacteriums：100 carry out being seeded to fresh 4mL LB trainings
It supports in base, is placed in 37 DEG C of constant-temperature tables, when rotating speed is that 250rpm/min cultivates 2~3h to OD600 values about 0.5 ± 0.1
It can；
3. after OD600 reaches prescribed requirement, bacterium solution is dispensed into 1.5mL EP pipes, often pipe 1mL, be placed in ice on ice
Bathe 10min；
4. the bacterium solution cooled down is placed in the refrigerated centrifuge of precooling, 4 DEG C, 4000rpm, after centrifugation 5min as far as possible
Abandon most supernatant；
5. the CaCl of the 0.1M of 1mL precoolings is added2Thalline, then ice bath 30min is resuspended；
6. 4 DEG C, 4000rpm, centrifuging and abandoning most supernatant as far as possible after 5min (dried filter paper will be residual after can using sterilizing
Liquid storage body exhausts)；
7. the CaCl of 50 μ L 0.1M is added2Thalline is resuspended, can be used after ice bath 0.5h, this is the DH5 α prepared
Competent cell；
8. the competence bacteria DH5 α prepared are placed on -20 DEG C to freeze and (used in one month).
(8) it converts
1. volume aspirated, which is the connection liquid of 2.5 μ L, is added to the DH5 α competent cells equipped with 50 μ L prepared in advance
EP pipes in, lightly blow and beat mixing with liquid-transfering gun, after blowing and beating mixing, be just positioned on ice, ice bath 30min；
2. after the completion of waiting for ice bath, it will be put in 42 DEG C of preheated in advance thermostat water baths added with the competence of connection liquid,
Heat shock 90s；
3. after completing heat shock, immediately competence is positioned on ice, ice bath 2min；
4. after ice bath 2min, the LB liquid medium of 500 μ L room temperatures is added into EP pipes, it is put in 37 DEG C of constant temperature oscillation trainings
Case oscillation is supported, rotating speed 200rpm cultivates 50min；
5. cultured bacterium is put in centrifuge, room temperature, 6000rpm centrifuges 1min, after centrifugation, carefully inhales
Supernatant is removed, about 100 μ L culture mediums are stayed, thalline is resuspended；
6. the thalline for drawing 100 μ L is coated on and pre-prepared has added ampicillin (final concentration of 100 μ g/mL)
It on the LB tablets of resistance, is placed in 37 DEG C of constant incubators, after just setting 15min, waits for that bacterium solution is absorbed completely, be inverted culture, training
It is 12~16h to support the time.
(9) double digestion is identified
1. second day after the completion of conversion, go out from picked clones on the LB tablets containing amicillin resistance after conversion
Single bacterium drops down onto 12~16h of culture in the LB liquid medium containing 100 μ g/mL ammonia benzyl resistances, is taken out by plasmid extraction kit
It is proposed recombinant plasmid.
2. the recombinant plasmid that extracting is obtained carries out double digestion identification, reaction system see the table below：
6 recombinant plasmid double digestion reaction system table of table
| Component | Volume (μ L) |
| 10×FastDigest Buffer | 2 |
| Recombinant plasmid dna | 6 |
| FastDigest enzyme Sal I | 1 |
| FastDigest enzyme Bamh I | 1 |
| ddH2O | 10 |
| Total volume | 20 |
Note：Endonuclease reaction condition：37 DEG C of constant temperature digestion 30min
3. double digestion product is taken pictures through 2% agarose gel analysis through gel imaging system, analyzes and preserve
As a result.
(10) recombinant plasmid Plasmid samples are sequenced
Send the Plasmid samples that can go out target gene size segment with double digestion to company's (raw work bioengineering (Shanghai) stock
Part Co., Ltd) sequencing, the UL146 gene orders that sequencing result is downloaded with GennBank are compared on NCBI.
(11) preparation of competence bacteria BL21 (DE3), with reference to (7).
(12) pET32a (+)-UL146 recombinant plasmid transformeds expression bacterium BL21 (DE3), with reference to (8).
Experimental result：The results are shown in Figure 3 for the double digestion of pET32a (+)-UL146 recombinant expression carriers.
Two, the prokaryotic expression of UL146 recombination fusion proteins
(1) the protokaryon induced expression temperature optimization of fusion protein
1. the cultured 200 μ L bacterium solutions of transferring are to the 150mL of the fresh 20mL LB containing 100 μ g/mL ammonia benzyl resistances
In conical flask, it is 0.5~0.8 to be put in 37 DEG C of shaken cultivations to OD600 values；
The IPTG derivants of final concentration of 1mM are added when 2. OD600 values are up to 0.5~0.8, be respectively placed in 20 DEG C, 30 DEG C,
It is cultivated in 37 DEG C of incubators, rotating speed 160rpm, oscillation induction 5h；
3. the bacterium solution 1mL before taking induction respectively and after induction, is placed in compact centrifuge, 6000rpm centrifuges 10min, abandons
Supernatant；
4. bacterial sediment is washed 3 times with 1mL PBS, 10000rpm centrifuges 5min, abandons supernatant；
5. 48 μ L PBS are added to be resuspended, and 5 × SDS-PAGE loading buffer are added, are placed in mediation oscillator
Mixing, 99 DEG C are boiled 5~10min, are immediately placed on cooled on ice 3min；
6. carrying out SDS-PAGE electrophoresis, expression product is separated by electrophoresis by 12% SDS-PAGE, in glue bottom about 1cm
Stop electrophoresis, 55V 50min, 120V about 40min when place.
PAGE gel electrophoresis step is as follows：
1. installing rectilinear electrophoresis tank；
2. preparing 12% separation gel (recombinant protein size is 35kD or so) see the table below：
7 12% separation gel of table is with tabulation
Between reagent is poured into glass plate after mixing, with water seal top, pay attention to that liquid level, gel is made to polymerize needs 30 completely
~60min；
3. 5% concentration glue of configuration see the table below：
Table 8 5% concentrates glue with tabulation
Water on separation gel is gone, filter paper is used in combination to blot excessive moisture, above-mentioned mixed liquor is added, immediately inserts comb
Between entering glass plate, polymerization completely needs 15~30min；
4. after spacer gel completely polymerization, comb is carefully extracted, then glass plate is fixed on electrophoresis tank；
5. loading；
6. electrophoresis：1 × electrophoretic buffer is added in electrophoresis tank, connects power supply, when electrophoresis, the voltage of spacer gel is set as
55V, separation gel voltage are set as 120V, and electrophoresis to bromophenol blue row to electrophoresis tank lower end stops (about needing 2~3h)；
7. dyeing：Glue is taken out from glass plate carefully, is put into coomassie brilliant blue staining liquid and dyes, it is placed at room temperature for 4~
6h。
8. decolourizing：Glue is taken out from dyeing liquor gently, is put into destainer, is placed on decolorization swinging table, is repeatedly decolourized extremely
Protein band is clear；
9. gel is imaged and is preserved：The gel for completing decoloration is imaged, if gel also has other experimental uses,
It can temporarily be stored in 7% acetic acid solution or distilled water.
Experimental result：SDS-PAGE analyzes the temperature optimization result of recombinant protein protokaryon induced expression as shown in Fig. 4, most
Good inducing temperature is 20 DEG C.
(2) the protokaryon induced expression inducer concentrations optimization of fusion protein
1. the cultured 300 μ L bacterium solutions of transferring are to the 250mL of the fresh 30mL LB containing 100 μ g/mL ammonia benzyl resistances
In conical flask, it is put in 37 DEG C of shaken cultivations and both may be used to OD600 values for 0.5~0.8；
Final concentration of 0.2mM is separately added into when 2. OD600 values are up to 0.5~0.8, the IPTG of 0.5mM, 1.0mM, 2.0mM are lured
Agent is led, is placed in 20 DEG C of incubators and cultivates, rotating speed 160rpm, oscillation induction 5h；
3. the bacterium solution 1mL before taking induction respectively and after induction, is placed in compact centrifuge, 6000rpm centrifuges 10min, abandons
Supernatant；
4. bacterial sediment is washed 3 times with 1mL PBS, 10000rpm centrifuges 5min, abandons supernatant；
5. 48 μ L PBS are added to be resuspended, and 5 × SDS-PAGE loading buffer are added, are placed in mediation oscillator
Mixing, 99 DEG C are boiled 5~10min, are immediately placed on cooled on ice 3min；
6. carrying out SDS-PAGE electrophoresis, expression product is separated by electrophoresis by 12% SDS-PAGE, in glue bottom about 1cm
Stop electrophoresis, 55V 50min, 120V about 40min when place.
Experimental result：SDS-PAGE analyzes IPTG concentration optimizations result such as Fig. 5 institutes of recombinant protein protokaryon induced expression
Show, best inducer concentrations are 0.5mM.
(3) the protokaryon induced expression of fusion protein is time-optimized
1. the cultured 300 μ L bacterium solutions of transferring are to the 250mL of the fresh 30mL LB for having ammonia benzyl resistance containing 100 μ g/mL
In conical flask, it is put in 37 DEG C of shaken cultivations and both may be used to OD600 values for 0.5~0.8；
It is separately added into the IPTG derivants of final concentration of 0.5mM when 2. OD600 values are up to 0.5~0.8, is placed in 20 DEG C of cultures
It is cultivated in case, rotating speed 160rpm, respectively oscillation induction 2h, 3h, 4h, 5h, 6h, 8 h, 10h, 12h, for 24 hours；
3. the bacterium solution 1mL before taking induction respectively and after induction, is placed in compact centrifuge, 6000rpm centrifuges 10min, abandons
Supernatant；
4. bacterial sediment is washed 3 times with 1mL PBS, 10000rpm centrifuges 5min, abandons supernatant；
5. 48 μ L PBS are added to be resuspended, and 5 × SDS-PAGE loading buffer, 12 μ L are added, are placed in mediation and shake
Mixing in device is swung, 99 DEG C are boiled 5~10min, are immediately placed on cooled on ice 3min；
6. carrying out SDS-PAGE electrophoresis, expression product is separated by electrophoresis by 12% SDS-PAGE, in glue bottom about 1cm
Stop electrophoresis, 55V 50min, 120V about 40min when place.
Experimental result：SDS-PAGE analyzes the time-optimized result of recombinant protein protokaryon induced expression as shown in Fig. 6, most
Good induction time is 10h.
(4) determination in the protokaryon induced expression place of fusion protein
1. the cultured 40 μ L bacterium solutions of transferring are to the test tube of the fresh 4mL LB containing 100 μ g/mL ammonia benzyl resistances
In, it is put in 37 DEG C of shaken cultivations and both may be used to OD600 values for 0.5~0.8；
It is separately added into the IPTG derivants of final concentration of 0.5mM when 2. OD600 values are up to 0.5~0.8, is placed in 20 DEG C of cultures
It is cultivated in case, rotating speed 160rpm, oscillation induction 10h；
3. the bacterium solution 1mL before taking induction respectively and after induction, is placed in compact centrifuge, 6000rpm centrifuges 10min, point
It does not obtain that front and back supernatant is induced to precipitate, supernatant is respectively placed in new 1.5mL EP pipes.In the front and back precipitation of induction
The PBS (pH=7.4) for being separately added into 4 DEG C of 1mL is resuspended, the thalline ultrasonication being resuspended.It is carried out using sonicator
Broken bacterium, broken bacterium parameter are set as：150W, work 5s stop 5s, are crushed 3~5min to bacterium solution clarification；
4. by the thalline re-suspension liquid being crushed with being flat in 4 DEG C of refrigerated centrifuges, 11000rpm, 4 DEG C centrifuge 30min,
Supernatant is taken to abandon precipitation；
5. taking 48 μ L of ready supernatant respectively, and 5 × SDS-PAGE loading buffer, 12 μ L are added, are placed in
Mixing in mediation oscillator, 99 DEG C are boiled 5~10min, are immediately placed on cooled on ice 3min；
6. a part is taken to carry out SDS-PAGE electrophoresis, expression product is separated by electrophoresis by 12% SDS-PAGE, at glue bottom
Stop electrophoresis, 55V 50min, 120V about 40min when at portion about 1cm；
7. remainder carries out Western Blot identifications.
Steps are as follows by Western Blot：
1. cell protein is detached through 12%SDS-PAGE gel electrophoresises first and is transferred on pvdf membrane；
2. the skimmed milk power room temperature with 5% closes 1h；
3. the pvdf membrane closed and anti-His mouse anti-(1:1000) it is incubated overnight altogether for 4 DEG C；
4. being washed 3 times with the TBST containing 1/2000 tween, each 20min；
5. the film after washing and fluorescence secondary antibody mountain sheep anti mouse (1:5000) it is incubated 2~3h altogether for 4 DEG C；
6. being washed 3 times with the TBST containing 1/2000 tween again, each 20min；
7. being exposed carry out immunoblotting assay finally by LI-COR ODYSSEY FC imaging systems.
Experimental result：The results are shown in Figure 7 in the expression place of SDS-PAGE analysis recombinant proteins, at the positions 35KD
It is recombinant protein to have obvious induced expression band, the band, and recombinant protein is in intracellular table as can be seen from the results
It reaches, and expression-form is inclusion body；The results are shown in Figure 8 in the expression place of Western blot identification recombinant proteins,
Western blot are the result shows that the albumen of induced expression is the recombinant protein containing UL146 genes, and further proof recombinates egg
Exist in the form of inclusion body in intracellular in vain.
(5) fusion protein ni-sepharose purification
1. transferring in cultured 4mL bacterium solutions to the conical flask of the fresh 2L containing 100 μ g/mL ammonia benzyl resistances, put
In 37 DEG C of shaken cultivations to OD600 values be 0.5~0.8；
It is separately added into the IPTG derivants of final concentration of 0.5mM when 2. OD600 values are up to 0.5~0.8, is placed in 20 DEG C of cultures
It is cultivated in case, rotating speed 160rpm, oscillation induction 10h；
3. collecting cultured thalline, with 8000rpm in 4 DEG C of high speed freezing centrifuges is flat on, 4 DEG C centrifuge 10min.
Abandon supernatant；
4. the bacterial sediment centrifuged is weighed, Wash 1buffer (50mM sodium phosphates, the 300mM of 10 times of volumes is added
Sodium chloride, 5mM imidazoles, 8M urea, pH 8.0) it is resuspended；
5. ultrasonication：55%, work 5s, stops 5s, is crushed 30min or so to thalline clarification；
6. by the thalline re-suspension liquid being crushed with 11000rpm in 4 DEG C of high speed freezing centrifuges is flat on, 4 DEG C centrifuge
30min abandons precipitation；
7. taking brokenly supernatant after bacterium, 0.22 μm of membrane filtration takes out 1mL and is placed in 4 DEG C of preservations.Remaining sample is placed in ice
It is upper to be purified；
8. filling column；
9. crossing column with the water of 2 times of column volumes (CV), the alcohol for preserving nickel column, columned rate 5mL/min are washed off；
10. column equilibration：It is balanced with the equilibrium liquid (50mM sodium phosphates, 300mM sodium chloride, pH 8.0) of 10CV, 2mL/min；
Loading, flow velocity 1mL/min collect column and wear；
Washing：It is first washed to baseline with the flow velocity of 1mL/min with Wash 1, collects column and wear；(the 50mM phosphorus of Wash 2 is used again
Sour sodium, 300mM sodium chloride, 25mM imidazoles, 8M urea, pH 8.0) foreign protein washed out to baseline with the flow velocity of 2 mL/min；
Elution：Eluent (50mM sodium phosphates, 300mM sodium chloride, 500mM imidazoles, 8M urea, pH 8.0) is with 2mL/
Min flow velocitys elute, and collect eluting peak；
Rebalancing：Equilibrium liquid is balanced with 2mL/min flow velocitys to baseline；
Storage：20% alcohol crosses column 5CV, 4 DEG C of freezer storage nickel columns；
By purified pool to protein solution and 7 in the non-purifying protein solution reserved respectively take 48 μ L, and be added 5 ×
12 μ L of SDS-PAGE loading buffer, are placed in mixing in mediation oscillator, and 99 DEG C are boiled 5~10 min, are immediately placed on
Cooled on ice 3min, remaining albumen after purification carry out dialysis desalination；
SDS-PAGE electrophoresis is carried out, expression product is separated by electrophoresis by 12% SDS-PAGE, in glue bottom about 1cm
Stop electrophoresis, 55V 50min, 120V about 40min when place.
Experimental result：The purification result of the recombinant protein of SDS-PAGE analyses after purification is as shown in figure 9, in the position of 35KD
There is band at the place of setting, which is destination protein, and purification result shows preferably purify mesh when imidazole concentration is 500 nM
Albumen, and analyze to obtain destination protein purity after purification up to 80% through gray scale scanning.
(6) protein sample is dialysed
1. bag filter is handled：The bag filter of 10cm long is placed in 500mL and contains 1mmol/L EDTA and 2%NaHCO3It is molten
10min is boiled in liquid, is taken out with clean tweezers or leather glove, then boils 10min in deionized water again, takes out dialysis
Bag is simultaneously i.e. usable after rinsed clean in deionized water or be placed in deionized water and save backup for 4 DEG C；
2. filling sample：Processed bag filter is taken, is first clamped one section with rubber band, protein sample is added in the other end, then
It is clamped with rubber band, the rubber band at bag filter both ends is tied up in a glass bar, bag filter frame on beaker, make somebody a mere figurehead both ends,
Prevent leaky protein；
3. dialysing：Deionized water is added toward beaker, regulates and bag filter is allowed to finish full bubble in deionized water as possible,
Beaker bottom puts a magneton, is placed on magnetic stirring apparatus and dialyses, deionized water is replaced after 30min, minimum to replace 15 times
Deionized water.
(7) content of BCA protein quantifications kit detection UL146 recombinant proteins
1. BSA Standard Solution are diluted to 500 μ g/mL using 1 × PBS；
2. BCASolution A and BCASolution B are pressed 50 according to sample number:1 dilution proportion at working solution,
It mixes well；
3. according to following table, the standard solution after dilution is configured in the sample well of 96 orifice plates：
The drafting of 9 BSA standard curves of table
4. after being diluted sample to be tested according to a certain percentage with 1 × PBS, 20 μ L is taken to be added in the sample well of 96 orifice plates；
5. the BCA working solutions of 200 μ L are added into sample well, 37 DEG C of placement 30min；
It is detected 6. 96 orifice plates are placed under 562nm wavelength；
7. drawing standard curve, testing protein sample concentration is calculated.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by change, modification, substitute, combination, letter
Change, should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL146 genes and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 357
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>UL146 genes
<400> 1
atgcgattaa tttttggttc gctgattagt cttttgatgg catttatgta ttaccatggg 60
gttcacagta gagaattgcg ttgtccgtgt acccacaaag ctttacatca tcctataggt 120
ggcttatttt gggttggtcg tgaccctccc aatcctcctg agtgtgacaa acctcaacat 180
tatctattgc ctcctcgagg taaacctgta tgtttagctc ccgatcatca tttgtccaaa 240
tggctggatg gtaaaaaaga caactcatgg cacagagtat tggtaaaagt aaaggatagt 300
aatggaccgc atgtagaaga aaacgctgtc actaacaaac gtccgcgttg gaaataa 357
<210> 2
<211> 118
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>UL146 albumen
<400> 2
Met Arg Leu Ile Phe Gly Ser Leu Ile Ser Leu Leu Met Ala Phe Met
1 5 10 15
Tyr Tyr His Gly Val His Ser Arg Glu Leu Arg Cys Pro Cys Thr His
20 25 30
Lys Ala Leu His His Pro Ile Gly Gly Leu Phe Trp Val Gly Arg Asp
35 40 45
Pro Pro Asn Pro Pro Glu Cys Asp Lys Pro Gln His Tyr Leu Leu Pro
50 55 60
Pro Arg Gly Lys Pro Val Cys Leu Ala Pro Asp His His Leu Ser Lys
65 70 75 80
Trp Leu Asp Gly Lys Lys Asp Asn Ser Trp His Arg Val Leu Val Lys
85 90 95
Val Lys Asp Ser Asn Gly Pro His Val Glu Glu Asn Ala Val Thr Asn
100 105 110
Lys Arg Pro Arg Trp Lys
115
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>UL146 primers-F
<400> 3
cgcggatcca tgcgattaat ttttggttcg ct 32
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>UL146 primers-R
<400> 4
acgcgtcgac ttccaacgcg gacgtttg 28
Claims (8)
1. a kind of recombinant plasmid containing human cytomegalovirus UL146 genes, it is characterised in that：Containing UL146 genetic fragments,
In, the nucleotide sequence such as SEQ ID No of UL146 genetic fragments：Shown in 1.
2. a kind of genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids, it is characterised in that：Conversion is had the right
It is required that the recombinant plasmid containing human cytomegalovirus UL146 genes described in 1.
3. the genetic engineering bacterium according to claim 2 containing human cytomegalovirus UL146 gene recombination plasmids, feature
It is：The genetic engineering bacterium is bacillus coli DH 5 alpha or BL21 (DE3).
4. the construction method of the genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids described in claim 2,
It is characterized in that：The recombinant plasmid transformed containing human cytomegalovirus UL146 genes described in claim 1 is entered into protokaryon table
In expression engineered bacteria, the genetic engineering bacterium containing human cytomegalovirus UL146 gene recombination plasmids is obtained.
5. the genetic engineering bacterium according to claim 2 or 3 containing human cytomegalovirus UL146 gene recombination plasmids is in UL146
Application in recombinant protein preparation.
6. a kind of preparation method of UL146 recombinant proteins, it is characterised in that：It will be big and small containing someone described in Claims 2 or 3
The genetic engineering bacterium of cellular virus UL146 gene recombination plasmids pass through activation culture and fermented and cultured, add derivant IPTG into
Row induced expression obtains UL146 recombinant proteins.
7. the preparation method of UL146 recombinant proteins according to claim 6, which is characterized in that further include that will obtain
The step of UL146 recombinant proteins are purified；
The purifying is to be purified using affinity chromatography.
8. the genetic engineering bacterium according to claim 2 or 3 containing human cytomegalovirus UL146 gene recombination plasmids is preparing people
Application in cytomegalovirus UL146 protein polyclone antibodies.
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|---|---|---|---|---|
| CN113788880A (en) * | 2021-09-12 | 2021-12-14 | 南京珀尔泰生物技术有限公司 | Purification method of human cytomegalovirus recombinant antigen |
| CN113788880B (en) * | 2021-09-12 | 2023-11-28 | 南京珀尔泰生物技术有限公司 | Purification method of human cytomegalovirus recombinant antigen |
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Application publication date: 20180803 |